Cholinergic receptor subtypes functional regulation in spinal cord injured monoplegic rats

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IN SPINAL CORD INJURED MONOPLEGIC RATS: EFFECT OF 5-HT, GABA AND BONE MARROW CELLS

THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

BIOTECHNOLOGY

UNDER THE FACULTY OF SCIENCE OF

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY

BY

CHINTHU ROMEO

DEPARTMENT OF BIOTECHNOLOGY

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY COCHIN - 682 022, KERALA, INDIA.

AUGUST 2012

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PROFESSOR

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DECLARATION

I hereby declare that the thesis entitled “Cholinergic receptor subtypes functional regulation in spinal cord injured monoplegic rats:

Effect of 5-HT, GABA and Bone Marrow Cells” is based on the original research carried out by me at the Department of Biotechnology, Cochin University of Science and Technology, under the guidance of Prof. C. S. Paulose, Director, Centre for Neuroscience, Department of Biotechnology and no part thereof has been presented for the award of any other degree, diploma, associateship or other similar titles or recognition.

Cochin - 682 022 Chinthu Romeo

17-08-2012 Reg. No. 3711

Department of Biotechnology Cochin University of Science and Technology

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ACKNOWLEDGEMENT ACKNOWLEDGEMENT ACKNOWLEDGEMENT ACKNOWLEDGEMENT

To the God God Almighty God God Almighty Almighty Almighty; the Creator and the Guardian, and to whom I owe my very existence. I bow before you, the merciful and the passionate, for providing me the opportunity to step in the excellent world of science. To be able to step strong and smooth in this way, I have also been supported and supervised by many people.

It is a pleasant moment to express my heartfelt gratitude to all.

I would like to extent my gratitude to Prof. C. S. Paulose, Prof. C. S. Paulose, Prof. C. S. Paulose, Prof. C. S. Paulose, Director, Centre for Neuroscience, Cochin University of Science & Technology for his supervision, advices, motivation and exemplary guidance throughout the course of my doctoral research. I extent my profound thankfulness, for his constant encouragement and timely advice. I am deeply grateful to him for providing me necessary facilities and giving me exposure to organizing skills which exceptionally inspired and enriched my growth as a student and a researcher as well. I am short of words to express my sincere appreciation for his patience and tolerance to me throughout this period. I solemnly submit my honest and humble thanks to him for bringing my dreams into reality. His understanding, encouraging and personal guidance have provided a good basis for the present thesis. I am solemnly indebted to him more than he knows or words can ever convey. His mentorship was paramount in providing a well rounded experience consistent my long-term career goals.

I offer my sincere thanks to Dr. E. Vijayan, ICMR Emeritus Professor,

Centre for Neuroscience, Dept. of Biotechnology, Cochin University of Science

and Technology and Dr. Oommen V Oommen, Department of Zoology, Kerala

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Marine Biology, Microbiology and Biochemistry, CUSAT for his help, encouragement and advice.

I would like to acknowledge Prof. M. Chandrasekaran, Dr. Sarita G.

Bhat, Dr. Padma Nambisan, faculties of the Department of Biotechnology, CUSAT, and Dr. Elyas K. K, former lecturer of our teachers of the Department for their advice and support throughout my work.

I take this opportunity to thank Prof. R. Muraleedharan Nair, Dean and Faculty of Science, Cochin University of Science & Technology for his help extended to me.

I thank all the teachers of my school days, graduation and post- graduation for laying my foundations. Especially, I thank Mr.R.Balabhaskar, Head of the Department, SRM Arts and Science college for his assistance and guidance in getting my post graduate career started on the right foot. He is one of the best teachers that I have had in my life

I sincerely acknowledge my senior colleagues Dr. Binoy Joseph, Dr.

Nair Amee Krishnakumar, Dr. Anu Joseph, Dr. Pretty Mary Abraham, Dr.

Jobin Mathew, Dr. Peeeyush Kumar T, Dr. Jes Paul and Dr Nandhu M S for their valuable suggestions and help.

I specially thank Dr. Sherin Antony and Dr. Anju T.R for all the support, love and motivation in all my efforts during the course of my work.

I am also obliged to my intimate friends Mrs. Anitha Malat and Mrs.

Shilpa Joy who were always with me, lending a helping hand and for their

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moral support. I also thank them all for the affection, love and friendship showered on me.

I am indebted to Mr. Naijil George for his selfless effort which greatly helped me in the completion of my research. Thanks to my junior Mr. Ajayan M S for his timely help and support during the later course of my research.

It is with immense pleasure I express my thankfulness to my batch mates Mr. Korah P Kuruvilla, Mr. Smijin Soman Mr. Jayanarayanan S and Ms. Roshni Baby Thomas. These friends and colleagues also provided for some much needed humor and entertainment in what could have otherwise been a somewhat stressful laboratory environment.

I also thank Ms. Jikku Jose, Ms. Anita Pinheiro, Mrs. Jasmine Koshy, Mrs. Ummu Habeeba, Mr. Raghul Subin S, Mr. Cikesh PC, Mr. Manzur Ali PP, Dr. Jissa G Krishna, Dr. Beena PS, Mr. P Karthikeyan, Mr. Sajan, Mr.

Siju M Varghese, Mrs. Jina Augustine, Mr. Satheesh Kumar M. K, Mrs.

Helvin Vincent, Mrs. Smitha S Mrs. Vijaya, Mrs. Sapna K,, Mr. Abraham Mathew, Mr. Ramesh Kumar S, Ms. Sudha Hariharan, Ms. Thresia Regimol TT and M.Sc. students of this Department for their friendship, help and co- operation. I also thank Mr. Abdul Rasheed A.P.

I thank the authorities of Amrita Institute of Medical Sciences and Research Centre, Cochin and Animal Breeding Centre, Kerala Agricultural University, Mannuthy for readily providing animals for this work.

This research has been supported and funded by various organizations

DST, KSCSTE, DBT, ICMR and CUSAT. I would like to extend my sincere

thanks to Cochin University of Science and Technology for supporting this

work by providing me with Junior and Senior Research Fellowship till

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thank the authorities and staffs of Cochin University of Science and Technology for their help and co-operation. I acknowledge my thanks to staff of Athulya Ladies Hostel of the University for their help and cooperation rendered during my stay.

Many friends have helped me stay sane through these difficult years.

Their support and care helped me overcome setbacks and stay focused on my study. I greatly value their friendship and I deeply appreciate their belief in me.

I wish to thank my dearest friends, Ms Liya Yesudas, Ms Anna Paul, Mr Mathew R Mathews, Mr Bibin Joy, Mr Sebastian Joy, Mr Jomy Jose and Mr Sandeep C for their friendship, love and relentless support in all my days of happiness and sorrows.

I also thank my college mates Mrs Aiswaraya, Mrs Revathy, Mrs Subashini, Mrs Prathibha, Mrs Emerald, Mr Imran, Mr prajith, Mr Karthik and Mr Mayur for their affection and encouragement. I thank them for their valuable suggestions. I specially thank Mrs Dhivya for her indespensable support, love and constant encouragement. I owe my thanks to Mr Ali Akbar for his friendship, love and care.

I thank Mrs Theja Sony for her love and informal support. I thank Mr

Roshan Thomas and Mr Jinesk K soman for their friendship and motivating me

in working and accomplishing my goal. I will never forget the times you all

have spent to make me lively and cheerful even in the cloudiest of my days.

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I thank Dr. George Mathew for his motivation, support and love. My special thanks to Dr.N.D.Inasu, former Pro.Vice Chancellor, CUSAT. I truly acknowledge them for all their help and guidance.

I owe my thanks to my dear husband Mr. Vivek Lawrence for giving me untiring support for the materialization of Doctoral study and for his unwavering love, quiet patience and pillar of support. His tolerance of my occasional bad moods is a testament in itself of his unyielding devotion and love. He has endured with aplomb and selflessness every emotion, all the fears and tears, and the countless hours of my solitude and detachment.

My joy knew no bounds in expressing the heartfelt thanks to my beloved parents Mr Romeo Verghese and Mrs Leena Romeo, my guiding lights, for their love, understanding, motivation and timely advise. Thank you both for giving me strength to reach for the stars and chase my dreams. They have been selfless in giving me the best of everything and I express my deep gratitude for their love without which this work would not have been completed. It was under their watchful eye that I gained so much drive and an ability to tackle challenges head on. I am deeply indebted to my dear brother Mr Joseph Romeo for his support.

I can barely find words to thank my dear papa and mummy- Mr. K G Lawrence and Mrs Valsa Lawrence for their words of wisdom, affection, endurance and constant support. They have been selfless in giving me the best of everything and I express my deep gratitude for their love without which this work would not have been completed. I warmly appreciate the generosity and understanding of my extended family. I express my loving thanks to Mr.

Pradeesh Lawrence, Mrs Julie Pradessh, Mr Praju and Ms Eliza for the jovial

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I remember thankfully the love and blessings showered upon me by my grandfathers Mr B C Verghese and Mr K J Louis and my grandmothers Mrs Rosy Verghese and Mrs Flower Louis for their love and prayers throughout my life. I would like to particularly acknowledge Mr Joseph Tenny, Mrs Sincy Tenny, Ms Agna and Mr Akarsh.

I specially thank Mr K G Felix and Mr Andrews for their inspiring words and support. I express my deep gratitude to Mrs Regina, Mrs Selina, Mrs Teresa Alex, Mrs Sheela Xavier, Mrs Sheela Felix, Mrs Leela Xavier, Mrs Lisa Andrews, Mrs Francina, Mrs Baby Mrs sany, Mrs Jessy and Mrs Rakhi Felix for their love and prayers.

I would like to thank Mrs Kunjumol Alex, Mr Unni, Mr Prashanth, Mr Nidhi, Mr Richu and Mr Ajun for their support and timely help. I aknowledge Mr. Elvis Paramel, Mr Rajendran vyrelil, Mrs Bindhu Rajendran and Mrs Sreedevi Sunil for their love and encouragement. I thank my beloved aunts, uncles and cousins for their support and prayers throughout my Doctoral study.

Finally, I would like to thank everybody who stood by me for the realization of this thesis from the bottom of my heart.

My tribute…..to a number of animals who have paid a price with their lives and suffering in the name of human protection. I pay my tribute to their sacrifice and pray that it is not in vain.

Chinthu Romeo

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Dedicated t Dedicated t Dedicated t Dedicated to o o o Lord Almighty Lord Almighty Lord Almighty Lord Almighty

The ultimate source of all knowledge and power

The ultimate source of all knowledge and power The ultimate source of all knowledge and power

The ultimate source of all knowledge and power . . . . . . . . . . . .

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5-HT Serotonin

ACh Acetylcholine

AChE Acetylcholine esterase

BDNF Brain-Derived Neurotrophic Factor

B_max Maximal binding

BMC Bone marrow cells

BrdU Bromodeoxyuridine

cAMP 3'-5'-cyclic adenosine monophosphate cGMP 3'-5'-cyclic guanosine monophosphate ChAT Choline acetyl transferase

CNS Central Nervous System

Cox Cyclo oxygenase

CREB cAMP regulatory element binding protein

CSF Cerebrospinal fluid

DAG Diacylglycerol

DNA Deoxy ribonucleic acid

DRG Dorsal Root Ganglion

EDTA Ethylene diamine tetra acetic acid

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GABA Gamma amino butyric acid GDNF Glial Derived Neurotrophic Factor GFR Glial cell line-derived neurotrophic factor

receptor

GPx Glutathione peroxidase

GSH Glutathione

IGF Insulin like growth factor

IL Inter leukine

IP₃ Inositol trisphosphate

iNOS inducible Nitric Oxide Synthase

K_d Dissociation constant

Mn SOD Manganese Superoxide Dismutase nAChRs Nicotinic acetylcholine receptors NeuN Neuronal-specific nuclear protein NF-κB Nuclear factor-kappa B

NGF Nerve growth factor

NTF Neurotrophic factors

PBS Phosphate buffered saline

PBST Phosphate buffered saline Triton X- 100

PCR Polymerase Chain Reaction

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PI3 Phosphatidyl inositol-3 PI3-K Phosphatidylinositol 3-kinase

PIP₂ Phosphatidylinositol 3,4-Bisphosphate PIP₃ Phosphatidylinositol 3,4,5-Trisphosphate

PKA Protein kinase A

PKC Protein kinase C

PLC Phospholipase C

QNB Quinuclidinylbenzilate

ROS Reactive oxygen species

SCI Spinal cord injury

SEM Standard error of mean

SOD Superoxide dismutase

TNF Tumour Necrosis factor

TNFR Tumour Necrosis factor receptor

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CONTENTS CONTENTS CONTENTS CONTENTS

INTRODUCTION 1

OBJECTIVES OF THE PRESENT STUDY 8

LITERATURE REVIEW 10

Current treatments and its side effects in SCI 13 Neurotransmitters and its receptors in SCI 15

5-HT 15

5-HT as co-mitogen 17

GABA 18

GABA as co-mitogen 20

Acetylcholine 22

Muscarinic Receptors 25

Muscarinic Receptors in SCI and regeneration 29 Nicotinic Acetyl choline Receptors (nAChR) 30 Nicotinic receptors in SCI and regeneration 34 Cholinergic enzymes – ChAT & AChE 35 Signal transduction through Second Messengers 36

Inositol 1,4,5-trisphosphate 37

3'-5'-cyclic guanosine monophosphate 38 3'-5'-cyclic adenosine monophosphate 39

Phospho Lipase C 39

cAMP regulatory element binding protein 40

Apoptosis & Spinal cord injury 41

Caspases 42

Bax 44

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activated B cells 46 Oxidative Stress and Spinal cord injury 48

Super oxide dismutase 50

Glutathione peroxidase 51

Neuronal Survival Factors in spinal cord injury 51

Brain Derived Neurotrophic Factor 53

Glial Derived Neurotrophic Factor 55

Insulin Like Growth Factor-1 56

Akt 58

Cyclins 60

Cell therapy in SCI 63

Bone marrow cells 63

MATERIALS AND METHODS 65

Chemicals used and their sources 65

Biochemicals 65

Radiochemicals 65

Molecular Biology Chemicals 65

Confocal Dyes 66

Animals 66

Experimental design 66

Treatment 67

Sacrifice and tissue preparation 68

BEHAVIOURAL STUDIES 68

Rotarod Test 68

Grid Walk Test 68

Narrow Beam Test 69

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MUSCARINIC RECEPTOR BINDING STUDIES

USING[³H] RADIO LIGANDS 69

Binding studies in the Spinal cord and brain regions 69 Total muscarinic, muscarinic M1 and M3

receptor binding studies 69

Protein determination 70

ANALYSIS OF THE RECEPTOR BINDING

DATA 70

Linear regression analysis for Scatchard plots 70 GENE EXPRESSION STUDIES IN SPINAL

CORD AND BRAIN REGIONS OF

CONTROL AND EXPERIMENTAL RATS 70

Isolation of RNA 70

REAL-TIME POLYMERASE CHAIN REACTION 71

cDNA synthesis 71

Real-time PCR assays 71

IP3 CONTENT IN THE SPINAL CORD AND BRAIN REGIONS OF CONTROL

AND EXPERIMENTAL RATS IN VIVO 73

Principle of the assay 73

Assay Protocol 73

cGMP CONTENT IN THE SPINAL CORD AND BRAIN REGIONS OF CONTROL

AND EXPERIMENTAL RATS IN VIVO 74

cAMP CONTENT IN THE BRAIN REGIONS OF CONTROL AND

EXPERIMENTAL RATS IN VIVO 76

Immuno histochemistry of Muscarinic m1, m3 and α7 nicotinic acetylcholine receptor in the

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Bone marrow cells differentiation studies

using BrdU AND NeuN 78

Statistics 79

RESULTS 80

Body weight of control and experimental rats 80

BEHAVIOURAL STUDIES 80

Rotarod performance of control and experimental rats 80 Behavioural response of control and experimental rats

on grid walk test 80

Behavioural response of control and experimental rats

on narrow beam test 81

SPINAL CORD 82

Total muscarinic receptor analysis 82

Scatchard analysis of [³H] QNB binding against atropine to total muscarinic receptor in the

spinal cord of control and experimental rats 82

Muscarinic M1 receptor analysis 82

Scatchard analysis of [³H] QNB binding against pirenzepine to muscarinic M1 receptor in the

Scatchard analysis of [³H] DAMP binding

against 4-DAMP mustard to muscarinic M3 receptor

in the spinal cord of control and experimental rats 83

REAL TIME-PCR ANALYSIS 83

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Real Time PCR analysis of acetylcholine esterase

in the spinal cord of control and experimental rats 83 Real Time-PCR analysis of choline acetyl transferase

in the spinal cord of control and experimental rats 84 Real Time-PCR analysis of muscarinic M1receptor

in the spinal cord of control and experimental rats 85 Real Time-PCR analysis of α7 nicotinic acetylcholine

receptor in the spinal cord of control and

experimental rats 85

Real Time-PCR analysis of phospholipase C receptor

in the spinal cord of control and experimental rats 86 Real Time-PCR analysis of CREB in the

spinal cord of control and experimental rats 86 Real Time-PCR analysis of Bax in the

spinal cord of control and experimental rats 86 Real Time-PCR analysis of caspase-8 in the

spinal cord of control and experimental rats 87 Real Time-PCR analysis of superoxide dismutase

in the spinal cord of control and experimental rats 87 Real Time-PCR analysis of glutathione peroxidase

in the spinal cord of control and experimental rats 88 Real Time-PCR analysis of TNFα_{in the}

spinal cord of control and experimental rats 88 Real Time-PCR analysis of NF-

κ

_{B in the}

spinal cord of control and experimental rats 88 Real Time-PCR analysis of BDNF in the

spinal cord of control and experimental rats 89 Real Time-PCR analysis of GDNF in the

spinal cord of control and experimental rats 89 Real Time-PCR analysis of IGF-1 in the

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Real Time-PCR analysis of cyclin D2 in the

spinal cord of control and experimental rats 90 IP3 content in the spinal cord of control

and experimental rats 90

cAMP content in the spinal cord of control

cGMP content in the spinal cord of control and

experimental rats 90

CONFOCAL STUDIES 91

Muscarinic M1 receptor antibody staining

in the spinal cord of control and experimental rats 91 Muscarinic M3 receptor antibody staining

in the spinal cord of control and experimental rats 92 α7 nicotinic acetylcholine receptor antibody staining

in the spinal cord of control and experimental rats 92 Brdu-NeuN co-labelling studies in the spinal cord of

control and experimental rats 93

CEREBRAL CORTEX 94

Total muscarinic receptor analysis 94

Scatchard analysis of [³H] QNB binding against atropine to total muscarinic receptor in the cerebral cortex

of control and experimental rats 94

Scatchard analysis of [³H] QNB binding against pirenzepine to muscarinic M1 receptor

in the cerebral cortex of control and experimental rats 94

Scatchard analysis of [³H] DAMP binding

against 4-DAMP mustard to muscarinic M3 receptor

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REAL TIME-PCR ANALYSIS 95 Real Time PCR analysis of acetylcholine esterase

in the cerebral cortex of control and experimental rats 95 Real Time-PCR analysis of choline acetyl transferase

in the cerebral cortex of control and experimental rats 95 Real Time-PCR analysis of muscarinic M1receptor

in the cerebral cortex of control and experimental rats 96 Real Time-PCR analysis of α7 nicotinic acetylcholine

receptor in the cerebral cortex of control

Real Time-PCR analysis of phospholipase C receptor

in the cerebral cortex of control and experimental rats 96 Real Time-PCR analysis of CREB in the cerebral

cortex of control and experimental rats 96

Real Time-PCR analysis of Bax in the cerebral

Real Time-PCR analysis of caspase-8 in the cerebral

Real Time-PCR analysis of superoxide dismutase

in the cerebral cortex of control and experimental rats 97 Real Time-PCR analysis of glutathione peroxidase

in the cerebral cortex of control and experimental rats 98 IP3 content in the cerebral cortex of

cAMP content in the cerebral cortex of

cGMP content in the cerebral cortex of

CONFOCAL STUDIES 98

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in the cerebral cortex of control and experimental rats 99 α7 nicotinic acetylcholine receptor antibody staining

CEREBELLUM 100

Total muscarinic receptor analysis 100

Scatchard analysis of [³H] QNB binding against atropine to total muscarinic receptor in the

cerebellum of control and experimental rats 100

Muscarinic M1 receptor analysis 100

Scatchard analysis of [³H] QNB binding against pirenzepine to muscarinic M1 receptor in the

cerebellum of control and experimental rats 100

Scatchard analysis of [³H] DAMP binding against muscarinic M3 receptor antagonist, 4-DAMP mustard

in the cerebellum of control and experimental rats. 100

REAL TIME-PCR ANALYSIS 101

Real Time-PCR analysis of acetylcholine esterase

in the cerebellum of control and experimental rats 101 Real Time-PCR analysis of choline acetyltransferase

in the cerebellum of control and experimental rats 101 Real Time-PCR analysis of muscarinic M1receptor

in the cerebellum of control and experimental rats 102

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Real Time-PCR analysis of α7 nicotinic acetylcholine

receptor in the cerebellum of control and experimental rats 102 Real Time-PCR analysis of phospholipase C

in the cerebellum of control and experimental rats 102 Real Time-PCR analysis of CREB in the cerebellum

of control and experimental rats 103

Real Time-PCR analysis of Bax in the cerebellum

Real Time-PCR analysis of caspase-8

in the cerebellum of control and experimental rats 103 Real Time-PCR analysis of superoxide dismutase

in the cerebellum of control and experimental rats 103 Real Time-PCR analysis of Glutathione peroxidase

in the cerebellum of control and experimental rats 103 IP3 content in the cerebellum of control

and experimental rats 103

cAMP content in the cerebellum of control

cGMP content in the cerebellum of control

CONFOCAL STUDIES 104

in the cerebellum of control and experimental rats 104 Muscarinic M3 receptor antibody staining

in the cerebellum of control and experimental rats 105 α7 nicotinic acetylcholine receptor antibody staining

in the cerebellum of control and experimental rats 105

BRAIN STEM 106

Scatchard analysis of [³H] QNB binding atropine to against total muscarinic receptor in the brain stem of

control and experimental rats 106

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pirenzepine to muscarinic M1 receptor

in the brain stem of control and experimental rats 106

Scatchard analysis of [³H] DAMP binding against 4-DAMP mustard to muscarinic M3 receptor

in the brain stem of control and experimental rats. 106

in the brain stem of control and experimental rats 107 Real Time-PCR analysis of choline acetyl transferase

in the brain stem of control and experimental rats 107 Real Time-PCR analysis of muscarinic M1receptor

in the brain stem of control and experimental rats 108 Real Time-PCR analysis of α7 nicotinic acetylcholine

receptor in the brain stem of control and experimental rats 108 Real Time-PCR analysis of phospholipase C

in the brain stem of control and experimental rats 108 Real Time-PCR analysis of CREB in the brain stem

Real Time-PCR analysis of Bax in the brain stem of

Real Time-PCR analysis of caspase-8 in the brain stem of

Real Time-PCR analysis of superoxide dismutase

in the brain stem of control and experimental rats 109 Real Time-PCR analysis of glutathione peroxidase

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IP3 content in the brain stem of control

cAMP content in the brain stem of control

cGMP content in the brain stem of control

in the brain stem of control and experimental rats 110 Muscarinic M3 receptor antibody staining

in the brain stem of control and experimental rats 111 α7 nicotinic acetylcholine receptor antibody staining

CORPUS STRIATUM 112

Scatchard analysis of [³H] QNB binding against total muscarinic receptor antagonist, atropine

in the corpus striatum of control and experimental rats 112

Scatchard analysis of [³H] QNB binding against muscarinic M1 receptor antagonist, pirenzepine

Scatchard analysis of [³H] DAMP binding against muscarinic M3 receptor antagonist, 4-DAMP mustard

in the corpus striatum of control and experimental rats 113 Real Time-PCR analysis of choline acetyl transferase

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Real Time-PCR analysis of muscarinic M2receptor

in the corpus striatum of control and experimental rats 114 Real Time-PCR analysis of muscarinic M3receptor

in the corpus striatum of control and experimental rats 114 Real Time-PCR analysis of α7 nicotinic acetylcholine

receptor in the corpus striatum of control

Real Time-PCR analysis of phospholipase C

in the corpus striatum of control and experimental rats 115 Real Time-PCR analysis of CREB in the corpus striatum

Real Time-PCR analysis of Bax in the corpus striatum

Real Time-PCR analysis of caspase-8in the

corpus striatum of control and experimental rats 115 Real Time-PCR analysis of glutathione peroxidase

in the corpus striatum of control and experimental rats 116 Real Time-PCR analysis of superoxide dismutase

in the corpus striatum of control and experimental rats 116 IP3 content in the corpus striatum of control

cAMP content in the corpus striatum of control

cGMP content in the corpus striatum of control

in the corpus striatum of control and experimental rats 117 Muscarinic M3 receptor antibody staining

in the corpus striatum of control and experimental rats 117 α7 nicotinic acetylcholine receptor antibody staining

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DISCUSSION 118

Body Weight 119

Motor deficits in SCI rats 119

Cholinergic enzyme alterations in

spinal cord and brain of control and experimental rats 121

Central Muscarinic Receptor Alterations 123

Spinal cord 123

Cerebral cortex 127

Cerebellum 129

Brain stem 131

Corpus striatum 133

α7 Nicotinic Receptor gene expression in spinal cord

and brain regions of control and experimental rats 135 Phospholipase C expression in Spinal cord and brain 137

CREB expression in Spinal cord and brain 139

Bax expression in Spinal cord and brain 141

Caspase-8 expression in Spinal cord and brain 142 Superoxide dismutase expression in spinal cord and brain 144 Glutathione peroxidase expression in spinal cord and brain 146

TNFα expression in Spinal cord 147

NF-κB expression in Spinal cord 148

BDNF expression in Spinal cord 150

GDNF expression in Spinal cord 151

IGF-1 expression in Spinal cord 152

Akt-1 expression in Spinal cord 154

Cyclin D2 expression in Spinal cord 155

Second messengers in spinal cord and brain regions 156 IP3 content in spinal cord and brain regions 157 cAMP content in spinal cord and brain regions 158 cGMP content in spinal cord and brain regions 160

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SUMMARY 163

CONCLUSION 173

REFERENCES LIST OF PUBLICATIONS

FIGURE LEGENDS

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Introduction Introduction Introduction Introduction

Spinal cord injury (SCI) is a major public health issue worldwide. It causes changes in all physical systems and functional abilities (Krause & Crewe, 1991). It is a devastating neurological injury, resulting in varying degrees of paralysis and sensory loss which are permanent and irreversible. In India, approximately fifteen lakh people are affected with SCI. Majority of them (82%) are males in the age group of 16-30 years (Gupta et al., 2008). A recent survey reported that the prevalence of SCI ranges from 236 per million in India to 1800 per million in the USA (Hagen et al., 2012). SCI is caused mainly from motor vehicle accidents, fall from heights, sports injuries (Dunn et al., 2000). It also results from a gunshot or knife wound that penetrates and damages spinal cord.

When the spinal cord is injured, the nerves above the level of the injury continue to work. However, below the level of the injury communication is disrupted which can result in loss of movement, sensation, bowel and bladder control. The spinal cord relay messages between the brain and various parts of the body. Disruption of the spinal cord leads to diminished transmission of descending control from the brain to motor neurons and ascending sensory information.

In medical terms, SCI is defined as “the occurrence of an acute, traumatic lesion of neural elements in the spinal canal resulting in temporary or permanent sensory deficit" (Thurman et al., 1995). An injury to the spinal cord occurs when pressure is applied to the spinal cord or the blood supply, which carries oxygen to the spinal cord is disrupted. The consequence of injury depends on the site of injury and completeness of injury. The higher the level of lesion, the greater is the injury. The major conditions that result from injury to the spinal cord are quadriplegia, paraplegia and monoplegia. Quadriplegia is the paralysis of all four limbs, hands and the trunk. Paraplegia involves paralysis from the chest or waist downwards. Monoplegia is the paralysis of one limb or hand. A complete injury

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results in no function below the level of injury, no sensation and no movement. An incomplete injury results in some functional disability below the level of injury.

The spinal cord nerve tissue is like brain tissue in that it usually does not fully recover when damaged.

The pathophysiology of SCI is characterized by an initial primary injury followed by secondary deterioration. SCI causes destruction of sensory nerve fibres and also lead to loss of sensation such as touch, pressure and temperature.

SCI often leads to a mutilation of the respiratory system (Beth et al., 2007), secondary musculoskeletal deterioration (Shields & Dudley-Javoroski, 2003) and sexual function. The neurologic symptoms include pain, numbness, paresthesias, muscle spasm, weakness and bowel/bladder changes. Other effects of SCI also includes low postural blood pressure (postural hypotension), inability to regulate blood pressure effectively, reduced control of body temperature (poikilothermic) and inability to sweat below the level of injury.

Since SCI affects CNS (central nervous system), its understanding lead to new strategies to reverse the damage caused by SCI. Gamma Amino Butyric Acid (GABA) (Todd et al., 1992), glycine (Todd & Sullivan, 1990), serotonin (5-HT) (Basbaum et al., 1978), norepinephrine (Dahlström & Fuxe, 1965), dopamine (Fleetwood-Walker & Coote, 1981), choline acetyl transferase (ChAT) (Todd, 1991), acetyl choline esterase (AChE) (Kása, 1986) are distributed throughout the spinal cord. There are reports suggesting that neurotransmitter release from intra spinal grafts is a highly relevant parameter to evaluate the functional ability of transplanted cells (Leanza et al., 1993b; Cenci et al., 1994; Leanza et al., 1999;

Cenci & Kalén, 2000).

Acetylcholine (ACh) is the key neurotransmitter for para sympathetic nervous system. It modulates spinal sensory processing in the dorsal horn (Myslinski & Randic, 1977; Urban et al., 1989) through the intrinsic cholinergic inter neurons found in the dorsal horn (Barber et al., 1984; Todd, 1991). ACh is also found in the motor neurons. (Villégier et al., 2010). Depletion in the motor

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Introduction

neurons causes a decrease in ACh concentration (Rosario et al., 2007). In mammals rhythmic limb movement, such as walking is controlled by pattern- generating neurons within the spinal cord. During early development, motor neurons seem to become spontaneously active and they release ACh, which excites neighbouring cells as a form of cell-cell communication. Motor neurons thus mediate locomotion via ACh. Knock off model of mice that lacked the enzyme necessary for synthesising ACh resulted in development of defective spinal circuit that lacked the control of leg movements. This demonstrates the relevance of ACh in control of leg movements. Thus ACh is necessary for a proper neural circuit.

Necrosis or cell death is a pathophysiological process that occurs as a result of secondary damage after SCI. Cell death continues to occur over several days and weeks following SCI. In the secondary phase, lipid peroxidation and free-radical production also occurs. The invading inflammatory cells increase the local concentrations of cytokines and chemokines. SCI triggers apoptosis, which kills oligodendrocytes in injured areas of the spinal cord days to weeks after the injury. Oligodendrocytes are the cells that form the myelin sheath around axons and speeds the conduction of nerve impulses. Apoptosis strips myelin from intact axons in adjacent ascending and descending pathways, which further impairs the spinal cords ability to communicate with the brain. Thus free radicals and apoptosis increase the damage in SCI. Both neurons and glia die by apoptosis; the response of oligodendrocytes in long tracts undergoing Wallerian degeneration is particularly long lived and is responsible for chronic demyelination and some of the dysfunction in chronic SCI. After SCI in the rat, posttraumatic necrosis occurred and apoptotic cells were found from 6 hours to 3 weeks after injury (Maria et al., 1997).

In the present scenario, basic research on SCI focuses on several areas that target functional restitution and regeneration of the injured neurons within the spinal cord. Stimulating the rejuvenation of axons is a key factor of spinal cord

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repair because every axon in the injured spinal cord that can be reconnected and increases the chances for improvement of function. Previously, CNS neurons were thought to be incapable of regeneration. But, Liu and Chambers (1958) indicated that central projections of primary afferent fibres can develop in the spinal cord after injury. Subsequent work by Richardson et al., (1980) demonstrated axonal elongation. Axonal growth alone is not sufficient for functional recovery. Axons have to make the proper connections and re-establish functioning synapses.

Therefore, SCI research should focus on preventing the loss of function and on restoring lost functions, including sensory and motor functions with the ultimate goal of fully restoring to the individual levels of activity and function that a person had before injury. Targets for intervention for improving functional outcome in SCI include free radical reduction, prevention of neuronal populations from apoptosis and promotion of neurite outgrowth.

With recent molecular strategies and techniques, research in the understanding of neuronal injury and neural regeneration provide new promises for reversal of SCI that was thought to be permanent and irrevocable (Carlson &

Gorden, 2002). A variety of tissues and cells have been implanted in the damaged spinal cord to restore function. These include bone marrow cells (BMCs), olfactory ensheathing cells, dorsal root ganglia, adrenal tissue, hybridomas, peripheral nerves or transplanted conduits of schwann cells. It is hypothesised that these cells would rescue, replace or provide a regenerative pathway for injured neurons, which would then integrate or promote the regeneration of the spinal cord and restore function after injury (Zompa et al., 1997). Thus the promising treatment of SCI is cell-based therapy (Stanworth & Newland, 2001; Hipp &

Atala, 2004) due to the limited success of pharmacological treatment. Cellular transplantation strategies have been used in various models of SCI (Eftekharpour et al., 2008). The cell replacement approach has the advantage that it promotes regeneration and repair. Regeneration involves replacement of lost or damaged neurons and induces axonal regeneration. Repair involves replacement of

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Introduction

supportive cells such as oligodendrocytes in order to prevent progressive demyelination and induce remyelination (Totoiu & Keirstead, 2005). In addition, BMC transplantation promotes protection of endogenous cells from further cell damage by attenuation of secondary injury process. BMC can also generate endoderm and ectoderm derivates including neural cells (Jiang et al., 2002; Kim et al., 2002). Non-embryonic sources of adult stem cells, which are not of ethical and legal concerns usually associated with embryonic stem cell research, offer great promise for the advancement of spinal cord treatment (Moore et al., 2006). BMCs support the repair of damaged tissues. Under specific experimental condition, BMCs differentiate into mature neurons or glial cells (Munoz et al., 2003).

Transplanted BMCs improvises neurological deficits in the CNS injury models by producing neural cells or myelin producing cells (Chopp et al., 2000; Akiyama et al., 2002). BMCs actively remyelinate spinal cord once administered directly or intravenously (Dezawa et al., 2005).

Consequences of SCI are devastating and any strategy to alleviate neurological loss is attractive (Treherne et al., 1992). Neurotransmitters relay, amplify and modulate signals between the neurons. 5-HT is known to play a facilitory role in locomotor circuit by increasing motoneuron excitability, modulating spinal central pattern generators (CPG) (Rossignol et al., 1988) and improving locomotor behaviour following SCI (Kim et al., 1999; Ribotta et al., 2000). A small amount of 5-HT can activate super-sensitive motor neurons (Li et al., 2007). Reports suggests that the cell-specific effect of 5-HT on regenerating neurons within the adult CNS by increasing the calcium concentration of the cells (Murrain et al., 1990). GABA receptors are known to be involved in during neuronal development. The presence of GABA receptors in developing oligodendrocytes provides a new mechanism for neuronal–glial interactions during development and offers a novel target for promoting remyelination following white matter injury (Luyt et al., 2007). GABA increases synaptic plasticity. Soltani et al., (2011) reported that GABA promotes proliferation of β-

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cells in pancreas. GABAergic inputs to hippocampal progenitor cells promote neuronal differentiation (Tozuka et al., 2005). Continuous application of GABA could promote dendritic growth in vivo, influence ganglion sensitivity to ACh and alter development of pre synaptic specialisation (Wolf et al., 1987). 5-HT and GABA can be also used as agents for cell proliferation and differentiation. Earlier reports from our lab showed that 5HT acting through specific receptor subtypes 5HT₂ (Sudha & Paulose, 1998) and GABA acting through specific receptor subtypes GABA_B (Biju et al., 2002) control cell proliferation and act as co- mitogens. These reports have paved way to study of the effect of 5-HT and GABA in after SCI.

SCI is a major cause of concern and the role of cholinergic neurotransmitter system in SCI has not been widely studied. In the present study, we have chosen Wistar rats as our model for SCI. Rats have been chosen to study not only because they are readily available but also because the morphological, biochemical and functional changes that occur are similar to those seen in humans (McTigue et al., 2000; Metz et al., 2000; Norenberg et al., 2004; Fleming et al., 2006). Since ACh is the major neurotransmitter in the motor neurons, the study of cholinergic alteration during SCI will enlighten the signalling pathways that is involved in the SCI mediated motor deficits. This study further investigated the effect of regenerative cell proliferation and differentiation in SCI, when BMCs, 5- HT and GABA are supplemented individually and in combination. The present study also investigated the second messenger alterations by studying inositol triphosphate (IP3), 3'-5'-cyclic adenosine monophosphate (cAMP) and 3'-5'-cyclic guanosine monophosphate (cGMP) functional regulation and gene expression of Phospholipase C (PLC) and cAMP regulatory element binding protein (CREB).

The changes in gene expression of anti oxidant enzymes like Superoxide dismutase (SOD) and Glutathione peroxidase (GPx) were investigated. Gene expression studies of apoptotic factors like Bax, Caspase-8, Tumour Necrosis factor α (TNFα) and Nuclear factor kappa-light-chain-enhancer of activated B

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Introduction

cells (NF-κB) were studied. The gene expression of neuronal survival factors Brain Derived Neurotrophic Factor (BDNF), Glial Derived Neurotrophic Factor (GDNF), insulin like growth factors (IGF-1), Akt-1 and Cyclin D2 were also studied. We also demonstrated the autologous differentiation of BMC to neurons using comitogenic 5-HT and GABA by confocal studies with Bromodeoxyuridine (BrdU) labelling and Neuronal-specific nuclear protein (NeuN) expression.

Behavioural studies were planned to evaluate the locomotor function in control and experimental rats. Our present study on 5-HT, GABA and BMC dependent regulation of muscarinic receptors in the spinal cord and brain will certainly enlighten novel therapeutic possibilities for the treatment of SCI.

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1. To induce spinal cord injury in rats and to study the effect of 5-HT, GABA and BMC treatment individually and in combination.

2. To investigate the behavioural changes in control and experimental rats using rotarod test, grid walk test and narrow beam test.

3. To study cholinergic receptors alterations and in the spinal cord and brain regions of control and experimental rats.

4. To analyse the muscarinic receptors - muscarinic M1, M2, M3, nicotinic receptor - α7 nAChR, cholinergic enzymes - AChE and ChAT gene expression in the spinal cord and brain regions of control and experimental rats using real time PCR.

5. To study gene expression studies of second messenger enzyme - PLC;

transcription factor - CREB; apoptotic factors - Bax, Caspase-8, TNFα and NF-κB; anti-oxidant enzymes SOD and GPX; cell survival factors - BDNF, GDNF, IGF-1, Akt-1 and Cyclin D2 in the spinal cord and brain regions of control and experimental rats using real time PCR.

6. To study the second messenger - IP3, cGMP and cAMP content in the spinal cord and brain regions of control and experimental rats.

7. To study the localization and expression status of muscarinic M1, M3 and α7 nAChR using confocal microscope by immunofluorescent specific

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antibodies in the spinal cord and brain sections of control and experimental rats using Confocal microscope.

8. To study neuronal regeneration using Brdu and NeuN in spinal cord using confocal microscope.

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Spinal cord injury is a devastating clinical problem that has permanent consequences. It has many medical, emotional and social consequences. It results in irreversible functional loss and life time disability (Sekhon & Fehlings, 2001).

SCI is seen mostly in younger age group people (O'Connor & Murray, 2005). In the western countries, it is estimated that about 5 per 100,000 people suffer from the disabilities caused by SCI. The reasons of SCI are vehicular accidents (44.8%), fall from heights (21.7%), acts of violence like gun shots (16%) and sports injuries (13%). Since 80% of cases occur in younger age group between the ages of 16 to 30 years, SCI causes a significant cost in terms of lifetime care and loss of productivity.

Damage to motor nerves results in paralysis or loss of control of movement. Damage to somatosensory nerves results in loss of sensation and perception; one can no longer feel touch, pain, temperature or be able to tell without looking where in space the nerve damaged body part is positioned. After injury, the spinal cord undergoes a series of pathologic changes, including micro haemorrhage, cytotoxic edema, neuronal necrosis, axonal fragmentation, demyelination, secondary cellular destruction and eventually cyst formation (Balentine, 1978; Balentine & Greene, 1984; Coutts & Keirstead, 2008). The most frequent neurological deficit associated with SCI is incomplete tetraplegia (30.6%), followed by complete paraplegia (25.8%), complete tetraplegia (22.1%) and incomplete paraplegia (19.3%).

An injury to the spinal cord affects the brain regions (Gomez et al., 2012).

When spinal cord is injured, there occurs a primary mechanical injury followed by a secondary injury. Primary injury is the initial mechanical damage, whereas secondary injury is progressive cell injury that begins in the gray matter and progresses into the white matter (Ballentine, 1978). Primary mechanical injury is

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caused by the direct compression of spinal cord. Primary injury mechanisms include acute compression, impact, missile and distraction forces. The severity and the site of injury determine the effect of primary injury. The primary mechanical injury disrupts axons, blood vessels and cell membranes. Damage to blood vessels can be toxic to the CNS (Asano et al., 1980). It results in pathogenesis.

The concept of secondary injury was first put forward by Allen (1911).

However, our knowledge of the exact mechanism through which primary injury triggers secondary injury is not very specific (Simon et al., 2009). The secondary injury phase involves vascular dysfunction, oedema, ischemia, excitotoxicity, electrolyte shifts, free radical production, inflammation and delayed apoptotic cell death. After SCI, the mammalian CNS fails to adequately regenerate due to intrinsic inhibitory factors expressed on central myelin and the extracellular matrix of the posttraumatic gliotic scar. Secondary injury disrupts the blood-spinal cord barrier and generates inflammatory response. Both barrier disruption and inflammation perturb the microenvironment and expose neurons to plasma- derived cells and molecules that can be injurious to intact, neighbouring tissue (Schlosshauer, 1993). Inflammation is considered to be an important element in secondary damage after SCI. This secondary damage leads to tissue loss and functional impairments. The immune responses are triggered by SCI and are mediated by a variety of factors that have both detrimental and beneficial effects.

Inflammation that results from secondary injury is characterized by the accumulation of activated microglia, macrophages and contributes to secondary pathogenesis (Blight, 1992; Hirschberg et al., 1994; Popovich et al., 1994; Blight et al., 1995; Bethea et al., 1998). Inflammatory cells are coupled with delayed neuronal death and demyelination (Blight, 1985; Davis et al., 1990; Dijkstra et al., 1994; Hirschberg et al., 1994). Therefore, strategies have focused on diminishing the secondary effects of SCI (Dumont et al., 2001; Hall & Traystman, 2009;

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Fehlings & Nguyen, 2010). SCI also leads to a range of alterations that are cytotoxic to both nerve cells and glial cells. Reports suggest that neurons and glial cells are changed permanently after SCI. Changes occur to segments above and below the SCI and these persistent changes lead to dysfunction. Hence, comprehension of secondary injury mechanisms and their complexities in SCI are invaluable requisite for planned therapeutic strategies: to stimulate axonal regrowth (regeneration), to arrest the self-perpetuating degeneration (neuroprotection), and the generation of new neurons and glia that will repopulate the site of injury and functionally integrate into the surviving neural tissue.

The most important physical consequences of a SCI are motor and sensory loss and impairments of bladder, bowel and sexual function leading to widespread disabilities in activities of daily life. There are certain health problems that arise secondary to the SCI. They are pain, spasms, pressure sores, urinary problems, bowel problems, respiratory failure, oedema and excessive sweating (Post et al., 1998). SCI is associated with respiratory complications. In case of acute SCI, 80% of cases are associated with respiratory complications (Tollefson

& Fondenes, 2012). The common respiratory complications are atelectasis, pneumonia and respiratory failure (Jackson & Groomes, 1994). Pulmonary dysfunction is the cause for the largest portion of morbidity after SCI (Fishburn et al., 1990; Linn et al., 2000). SCI causes instant damage of nervous tissue followed by the loss of motor and sensory function. Due to the restricted self-repair ability of damaged nervous tissue, there underlies the need for reparative interventions to restore function after SCI. Without control from the brain, movements produced by a spinal CPG were not likely to be useful in restoring successful walking without regulation from the brain.

Magnetic Resonance Imaging (MRI) is the method to evaluate patients who have a persistent neurological deficit following SCI as it allows direct visualization of the injured cord, bony intervertebral and ligamentous structures,

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and paraspinal soft tissues. MRI has replaced myelography and Computer Tomography myelography as the primary imaging preference available to assess compression of the spinal cord and is also a vital diagnostic modality in cases of SCI without radiographic abnormality. MRI also provides information regarding prognosis and neurological recovery.

CURRENT TREATMENTS AND ITS SIDE EFFECTS IN SPINAL CORD INJURY

There are various treatments available for SCI. Corticosteroids are used for the pharmacological treatment of SCI. They act by improving the blood flow in the spinal cord, restore impulse transmission, regulates calcium metabolism and enhance functional neurological recovery (Anderson et al., 1982; Bracken, 1992;

Hall, 1993; Constantini, 1994). Methyl prednisolone is a cortico steroid that has anti-oxidant activity and is used in SCI treatment (Bracken, 1990). A study by Yu et al., (2004) reported that early repeated methyl prednisolone sodium succinate treatment allows greater recovery from SCI. Sharma et al., (2004) suggested that methyl prednisolone sodium succinate was of use in promoting post traumatic clinical recovery when given 1h after trauma. Methyl prednisolone sodium succinate proved to be more effective than dexamethasone in reducing edema when both are given after an interval of 1h (Sharma et al., 2004).

Methylprednisolone also has a neuro protective effect (Amar & Levy, 1999). It improves neurologic function when given within 8 hours after injury (Bracken et al., 1990). It also has various side effects. It causes severe allergic reactions (rash, hives, itching, difficulty breathing, tightness in the chest, swelling of the mouth, face, lips, or tongue, unusual hoarseness) bloody black or tarry stools, changes in body fat, chest pain, fainting, fever, chills or sore throat, increased hunger, thirst or urination, mental or mood changes (eg, depression, personality or behavioural changes), muscle pain, weakness or wasting, seizures, severe nausea or vomiting, shortness of breath, slow fast or irregular heartbeat, slow wound healing, stomach

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pain, sudden severe dizziness or headache, swelling of the feet or legs, tendon bone or joint pain, thinning or discoloration of the skin, unusual bruising or bleeding, unusual skin sensation, unusual weight gain, vision changes or other eye problems and vomit that looks like coffee grounds.

Lazeroids lack gluco corticoid activity and inhibits free radical formation.

They also inhibit lipid peroxidation and arachidonic acid formation (Quarles et al., 1990). Endogenous opioids, acting through opiate receptors within the spinal cord, mediate certain secondary pathophysiological changes that contribute to irreversible tissue injury. Opiate receptor antagonists also reduces the secondary damage that occurs after SCI (Faden & Salzman, 1992). Hyperglycaemia increases reactive acidosis and triggers biochemical events such as increase in calcium levels and break down of cell membrane that lead to neuronal death (Sala et al., 1999). Calcium plays a key role in neuronal injury. Therefore calcium channel blockers are used to treat SCI. Nimodipine is one such calcium channel blocker that increases the rat spinal cord blood flow. Free radical scavengers and anti-oxidants are also used to treat SCI (Hall, 1992). GM1 Ganglioside, a complex acidic glycolipid compound present in the neuronal membrane is also used in the treatment of SCI (Geisler et al., 1991). Adenosine also has a neuro protective effect on SCI (Sulfianova et al., 2002). Levetiracetam prolonged the survival and the function of spinal motor neurons and have a therapeutic potential for several diseases that kill or degenerate the spinal motor neurons, including SCI (Yasuhiro et al., 2012).

Clinically existing treatments provide modest benefit; therefore present research is aimed at developing more effective therapies for spinal cord repair and regeneration (Kwon et al., 2004; Baptiste & Fehlings, 2007; Ali & Bahbahani, 2010; Fehlings & Nguyen, 2010). None of the human trials has produced a major progress in neurological recovery or a meaningful increase in function (Tator, 2006; Simon et al., 2009; Wang et al., 2009; Jablonska et al., 2010). Cell-based

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strategies to remyelinate spared axons is an attractive emerging approach in the treatment of SCI.

NEUROTRANSMITTERS AND ITS RECEPTORS IN SPINAL CORD INJURY

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. Neurons within the spinal cord represent a primary site for the integration of somatosensory input. Spinal sensory integration is a dynamic process regulated by factors that include multisensory convergence and pathway selection (Lundberg, 1979; Baldissera et al., 1981;

Jankowska, 1992). The transmission of the sensory information begins with activation of the peripheral receptors of primary afferent neurons whose cell bodies lie within the dorsal root ganglia (DRG) and whose central terminals project to secondary neurons in the dorsal horn of the spinal cord. Several neurotransmitters and a large variety of receptors have been found in the superficial laminae of the dorsal horn. Transmission of the somatosensory information from the primary afferent fibers to the secondary dorsal horn neurons depends on the balance between the excitatory effects of excitatory amino acids and the inhibitory actions of several other transmitter systems. Neurotransmitter signalling has profound influence on the normal sequence of events involved in development of the spinal cord and hence locomotion. Neurotransmitters that promote cell proliferation include ACh, 5-HT, GABA.

5-HT

5-HT is present in the axons and terminals of raphe-spinal neurons in the dorsal horn, especially in the superficial laminae, laminae I-III. The origin of serotonergic projection to the dorsal horn is mainly the nucleus raphe magnus (Dahlström & Fuxe, 1965; Fuxe, 1965; Basbaum et al., 1978; Miletic et al., 1984).

5-HT and several peptides may be co-localized in the same raphe neurons and in

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their terminals. 5-HT may also be co-localized with GABA (Millhorn et al., 1987a,b). Molecular cloning has identified seven distinct families of 5-HT receptors (5-HT1-7). The 5-HT3 family consists of ligand gated ion channel receptors. The other 6 families interact with G-proteins and are coupled to second messengers. Three 5-HT receptor subtypes influence the dorsal horn somatosensory processing: 5-HT1, 5-HT2 and 5-HT3. There are three major sources of 5-HT receptors to the spinal cord dorsal horn: the DRG cells, the intrinsic spinal neurons and the descending systems. Neonatal capsaicin treatment or dorsal rhizotomy decrease 5-HT1A and 5-HT3 receptor binding in laminae I and II, but some still remains, indicating both pre and post synaptic localizations. A large majority of the 5-HT receptors in the dorsal horn do not participate in classic synapses, but are found in extra synaptic sites along the dendrites and somas.

The 5-HT systems are widespread throughout the brain, with most of the cell bodies of serotonergic neurons located in the raphe nuclei of the midline brain stem (Palacios et al., 1990). The largest collections of 5-HT neurons are in the dorsal and median raphe nuclei of the caudal midbrain (Jacobs & Azmitia, 1992).

The neurons of these nuclei project widely over the thalamus, hypothalamus, basal ganglia, basal forebrain and the entire neocortex. Interestingly, these 5-HT neurons also provide a dense subependymal plexus throughout the lateral and third ventricles. Activation of this innervations result in 5-HT release into the cerebrospinal fluid (CSF) and measurement of 5-HT content in CSF in disease states will largely reflect this pool (Chan-Palay, 1976).

The activation of 5-HT receptors can produce multiple physiological events, as 5-HT receptor families can either promote or inhibit different second messenger systems. Intrathecally administered 5-HT can either inhibit or stimulate (Hylden & Wilcox, 1983; Clatworthy et al., 1988) nociceptive reflexes.

Iontophoretic application in the vicinity of dorsal horn neurons generally causes inhibition (Griersmith & Duggan, 1980), although excitatory effects have also